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| | Cell-Free Assay for Evaluating Inhibition of ALK5 Kinase Phosphorylatio |
| Description: | ALK5 protein was expressed in Sf9 insect cells as human recombinant GST-fusion protein using the baculovirus expression system. Expressed protein was purified by affinity chromatography using GSH-agarose (Sigma-Aldrich). Kinase assay was performed in 96-well FlashPlates from Perkin Elmer (Boston, Mass., USA) in a 50 μL reaction volume. The reaction cocktail was pipetted in four steps in the following order: 20 μL of assay buffer (standard buffer), 5 μL of ATP solution in H2O, 5 μL of each test compounds of formula (I) in 10% DMSO, 10 μL of GSK3 (14-27) (200 ng)/10 μL of ALK5 solution (1 ng) (premixed). The reaction cocktail contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 Na3VO4, 1.2 mM DTT, 50 μg/mL PEG20000, 1 μM [γ-33P]-ATP (approximately 2.5×105 cpm per well), 200 ng/10 μL GSK3 (14-27), and 1 ng/10 μL ALK5. The reaction cocktail was incubated at 30° C. for 60 min. The reaction was stopped with 50 μL of 2% (v/v) H3PO4, and plates were aspirated and washed two times with 200 μL 0.9% (w/v) NaCl. Assay was performed with a BeckmanCoulter Biomek 2000 robotic system. Incorporation of 33Pi (counting of cpm) was determined with a microplate scintillation counter (Microbeta, Wallac). |
| Affinity data for this assay | |
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